DDAO compounds as fluorescent reference standards

ABSTRACT

According to the present teachings, methods and compositions are provided that utilize at least one reference dye of formula (I): 
                         
In some embodiments, a method comprises measuring a detection signal of a reporter dye and at least one reference dye of formula (I). In some embodiments, a composition comprises a reference dye of formula (1), a buffer, a selection of nucleotides and a protein.

BACKGROUND

A reference dye can be beneficial in many different many assays.Reference dyes can be used for calibration, normalization, and to ensurean assay is being carried out properly. In multiplex reactions, whichmay contain multiple dyes to identify multiple targets, it can bedifficult to find a stable reference dye that emits at a wavelengthdistinct from the other dyes and does not interfere with the reaction.

SUMMARY

In some embodiments, a method is provided, wherein the method maycomprise (a) forming a mixture comprising at least one probe and areference dye, wherein each of the at least one probes comprises atarget-specific moiety and a reporter dye, wherein each target-specificmoiety is specific for a different target and each reporter dye isdifferent from the others, and wherein the reference dye has formula(I):

-   -   wherein:    -   each of R₁ to R₃ and R₆ to R₈ is independently —H, halogen,        —CO₂H, —CO₂R, —SO₃H, —SO₃R, —CH₂CO₂H, —CH₂CO₂R, —CH₂SO₃H,        —CH₂SO₃R, —CH₂NH₂, —CH₂NHR, —NO₂, C₁-C₆ alkyl, substituted C₁-C₆        alkyl, C₁-C₆ alkoxy, and substituted C₁-C₆ alkoxy, wherein R is        C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, and        substituted C₁-C₆ alkoxy;    -   R₄ and R₅ taken separately are selected from C₁-C₆ alkyl, and        C₁-C₆ substituted alkyl, or R₄ and R₅ taken together are        selected from C₃-C₇ cycloalkyl, C₄-C₇ unsaturated cycloalkyl,        C₃-C₇ substituted cycloalkyl, or C₄-C₇ substituted unsaturated        cycloalkyl; and        (b) measuring fluorescence emitted by the reference dye; (c)        measuring fluorescence emitted by and at least one reporter dye.        In various embodiments, the method may additionally include        adjusting the measured fluorescence emitted by the at least one        reporter dye based on the measured fluorescence emitted by the        reference dye, to form an adjusted measurement. In some        embodiments, the mixture comprises at least two, at least three,        at least four, or at least five probes.

In some embodiments, R₁ is selected from hydrogen, halogen, methyl, andethyl; R₂ and R₃ are each independently a halogen; R₄ and R₅ are eachindependently selected from methyl and ethyl; R₆ is selected fromhydrogen, halogen, methyl, and ethyl; R₇ is selected from hydrogen,halogen, methyl, ethyl, and SO₃H; and R₈ is selected from hydrogen,halogen, methyl, and ethyl. In some embodiments, R₂ and R₃ are eachchlorine and R₇ is hydrogen or SO₃H. In some embodiments, the referencedye is selected from:

In some embodiments, the target-specific moiety may be selected from anoligonucleotide, a peptide, an antibody, an antigen, a small molecule, ametabolite, and a polysaccharide. In some embodiments, the target may beselected from a polynucleotide, an antigen, an antibody, a receptor, anenzyme, an organelle, a membrane, and a cell. In some embodiments, thetarget-specific moiety may be an oligonucleotide. In some embodiments,the probe comprises an oligonucleotide, a reporter dye, and a quencherdye. In some embodiments, the target may be a polynucleotide. In someembodiments, the method may comprise amplifying at least a portion ofthe polynucleotide.

In some embodiments, the fluorescence emitted by each reporter dye canbe measured in the presence of the other reporter dyes and the referencedye in the mixture. In some embodiments, the fluorescence emitted by thereference dye can be measured in the presence of the reporter dyes inthe mixture.

In some embodiments, a method may comprise: irradiating the mixture witha first excitation wavelength range; detecting radiation emitted by atleast a first reporter dye; irradiating the mixture with a secondexcitation wavelength range that differs from the first excitationwavelength range; and detecting radiation emitted by the reference dye.In some embodiments, a method may further comprise: irradiating themixture with a third excitation wavelength range; and detectingradiation emitted by at least a second reporter dye.

In some embodiments, a method may comprise: irradiating the mixture witha first excitation wavelength range; detecting radiation emitted by atleast a first reporter dye; and detecting radiation emitted by at leasta second reporter dye; wherein the radiation emitted by at least a firstreporter dye can be detected separately from the radiation emitted by atleast a second reporter dye.

According to various embodiments, a method can further compriseirradiating the nucleic acid-containing sample with a first excitationwavelength range, detecting radiation emitted from at least one dye of aplurality of dyes upon irradiation of the sample with the firstexcitation wavelength range, irradiating the sample with a secondexcitation wavelength range that differs from the first excitationwavelength range, and detecting radiation emitted from at least oneother of a plurality of dyes upon irradiation of the sample with thesecond excitation wavelength range. A different wavelength range forexcitation can be used for each different reporter dye of the pluralityof dyes, and for the passive reference dye.

In some embodiments, a composition may be provided, wherein acomposition comprises a master mix utilizing a reference dye of formula(I):

wherein:

-   -   each of R₁ to R₃ and R₆ to R₈ is independently —H, halogen,        —CO₂H, —CO₂R, —SO₃H, —SO₃R, —CH₂CO₂H, —CH₂CO₂R, —CH₂SO₃H,        —CH₂SO₃R, —CH₂NH₂, —CH₂NHR, —NO₂, C₁-C₆ alkyl, substituted C₁-C₆        alkyl, C₁-C₆ alkoxy, and substituted C₁-C₆ alkoxy, wherein R is        C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, and        substituted C₁-C₆ alkoxy;    -   R₄ and R₅ taken separately are selected from C₁-C₆ alkyl, and        C₁-C₆ substituted alkyl, or R₄ and R₅ taken together are        selected from C₃-C₇ cycloalkyl, C₄-C₇ unsaturated cycloalkyl,        C₃-C₇ substituted cycloalkyl, or C₄-C₇ substituted unsaturated        cycloalkyl.

In some embodiments, R₁ is selected from hydrogen, halogen, methyl, andethyl; R₂ and R₃ are each independently a halogen; R₄ and R₅ are eachindependently selected from methyl and ethyl; R₆ is selected fromhydrogen, halogen, methyl, and ethyl; R₇ is selected from hydrogen,halogen, methyl, ethyl, and SO₃H; and R₈ is selected from hydrogen,halogen, methyl, and ethyl. In some embodiments, R₂ and R₃ are eachchlorine and R₇ is SO₃H. In some embodiments, the reference dye isselected from:

According to various embodiments, a master mix may contain a buffer, aselection of nucleotides, for example, but not limited bydeoxynucleotides (dNTPS i.e. dATP, dGTP, dCTP, and TTP), primers, and atleast one protein moiety. In various embodiments, a master mix maycontain a buffer, a selection of nucleotides, for example, but notlimited by, dNTPS (i.e. dATP, dGTP, dCTP, and TTP), primers, at leastone protein moiety, and a reference dye. In various embodiments, amaster mix may contain a buffer, a selection of nucleotides, forexample, but not limited by, dNTPS (i.e. dATP, dGTP, dCTP, and TTP), andat least one protein moiety. In various embodiments, a master mix maycontain a buffer, a selection of nucleotides, for example, but notlimited by, dNTPS (i.e. dATP, dGTP, dCTP, and TTP), at least one proteinmoiety, and a reference dye. In various embodiments, a master mix may besupplied lyophilized or suspended in a buffer solution.

In some embodiments, a kit may include a reference dye of formula (1).In some embodiments, the reference dye may be an ingredient in a mastermix in a kit. In some embodiments, the reference dye and a master mixmaybe separate containers in a kit. In some embodiments, the referencedye and each of the at least one probes may be in separate containers ina kit. In some embodiments, the reference dye may be in a master mix,and the master mix and each of the at least one probes are in separatecontainers in a kit. In some embodiments, the reference dye, a mastermix, and each of the at least one probes may be in separate containersin a kit. In some embodiments, the reference dye and at least one of theprobes may be in the same container in the kit. In some embodiments, atleast two of the probes may be in the same container, while thereference dye is in a separate container in the kit. In variousembodiments, the kit components may be supplied lyophilized or suspendedin a buffer solution.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram of a PCR instrument according to variousembodiments of methods of the present teachings.

FIG. 2 is a block diagram of a PCR instrument according to variousembodiments of methods of the present teachings.

FIG. 3 is a block diagram that illustrates components of an exemplarycomputer system that may be utilized in the control and interface of thePCR instrumentation according to various embodiments of methods of thepresent teachings.

FIG. 4 is a graph showing the fluorescence emission and excitationspectra of DDAO, which may be used as a reference dye according tovarious embodiments of the present teachings.

FIG. 5 is a graph showing the fluorescence emission and excitationspectra of Sulfo-DDAO, which may be used as a reference dye according tovarious embodiments of the present teachings.

FIG. 6 is a graph showing the fluorescent emission signal detected as afunction of temperature for Sulfo-DDAO in comparison to two other dyes.

FIG. 7 is a graph showing the chemical stability of reference dyesaccording to the present teachings.

FIG. 8 is a matrix of different mixtures of four reporter dye-labeledoligonucleotides and Sulfo-DDAO reference dye, which were mixed togetherin various concentrations to simulate different possible end-pointreadings for genotyping assay signals, and to test the ability ofSulfo-DDAO to be used as a reference dye under each of the sixteendifferent simulated conditions.

FIGS. 9A and 9B show scatter plots of fluorescence signal intensitiesgenerated using pure dye mixtures from FIG. 8. FIG. 9A is a plot ofreporter dyes FAM® and VIC® emissions, which were deconvolved from thebackground of reporter dyes TED® and SID® using Sulfo-DDAO as areference dye. FIG. 9B is a plot of reporter dyes TED® and SID®emissions, which are deconvolved from the background of reporter dyesFAM® and VIC® using Sulfo-DDAO as a reference dye

FIGS. 10A and 10B show scatter plots of fluorescence signal intensitiesgenerated using pure dye mixtures from FIG. 8. FIG. 10A is a plot ofreporter dyes FAM® and VIC® emissions, which were deconvolved from thebackground of reporter dyes TED® and SID® using Sulfo-DDAO as areference dye. FIG. 10B shows the inability to deconvolve reporter dyesFAM® and VIC® emissions in a background of reporter dyes TED® and SID®,using a ROX reference dye.

FIG. 11 is a graph showing the chemical stability of Sulfo-DDAO, whenused as a reference dye in a universal master mix, measured over afour-week period as measured by assay activity, and in the presence of avariety of different types of probes.

FIG. 12 shows the normalized fluorescence emission spectra of each dyeof a set of dyes according to various embodiments of the presentteachings, wherein the set includes Sulfo-DDAO as a reference dye andfour reporter dyes of shorter emission wavelengths.

FIG. 13 shows a graph of normalized fluorescence emission spectra for aset of dyes according to various embodiments of the present teachings,wherein the set comprises Sulfo-DDAO as a reference dye and fourreporter dyes of shorter emission wavelengths.

FIG. 14 shows a graph of normalized fluorescence emission spectra for aset of dyes according to various embodiments of the present teachings,wherein the set comprises Sulfo-DDAO as a reference dye and fourreporter dyes of shorter emission wavelengths.

FIG. 15 is a graph showing the normalized fluorescence emission spectraof each dye of a set of six dyes according to various embodiments of thepresent teachings, wherein the set includes Sulfo-DDAO as a referencedye and five reporter dyes of shorter emission wavelengths.

FIG. 16 is a graph showing the normalized fluorescence emission spectraof each dye of a set of seven dyes according to various embodiments ofthe present teachings, wherein the set includes Sulfo-DDAO as areference dye and six reporter dyes of shorter emission wavelengths.

FIG. 17 is a matrix showing an optimized use of Sulfo-DDAO as areference dye in a four-reporter TAQMAN® Duplex SNP Genotypingexperiment wherein about 98% of the reaction sets yielded goodgenotyping results.

DETAILED DESCRIPTION

According to various embodiments of methods and compositions of thepresent teachings, a reference dye is provided, wherein the referencedye is of formula (I):

wherein:

-   -   each of R1 to R3 and R6 to R8 is independently —H, halogen,        —CO2H, —CO2R, —SO3H, —SO3R, —CH2CO2H, —CH2CO2R, —CH2SO3H,        —CH2SO3R, —CH2NH2, —CH2NHR, —NO2, C1-C6 alkyl, substituted C1-C6        alkyl, C1-C6 alkoxy, and substituted C1-C6 alkoxy, wherein R is        C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, and        substituted C1-C6 alkoxy;    -   R4 and R5 are either, taken separately, independently selected        from a C1-C6 alkyl and a C1-C6 substituted alkyl, or, taken        together, are C3-C7 cycloalkyl, C4-C7 unsaturated cycloalkyl,        C3-C7 substituted cycloalkyl, or C4-C7 substituted unsaturated        cycloalkyl.

As used herein, “substituted” refers to a molecule wherein one or morehydrogen atoms are replaced with one or more non-hydrogen atoms,functional groups or moieties. For example, unsubstituted amine is —NH₂,while on-limiting exemplary substituted amines include, but are notlimited to, —NHCH₃ and —N(CH₂CH₃)₂. Similarly, an exemplaryunsubstituted alkyl is —CH₂CH₃, while on-limiting exemplarycorresponding substituted alkyls include, but are not limited to,—CH₂CH₂COOH, —CH(NH₃)CH₃, and —CH═CHCOOCH₃. Exemplary substituentsinclude, but are not limited to, halogen, fluorine, chlorine, bromine,C₁-C₆ alkyl, C₁-C₆ cycloalkyl, C₁-C₆ branched alkyl, C₁-C₆ alkene, C₁-C₆cyclic alkene, C₁-C₆ branched alkene, C₁-C₆ alkyne, C₁-C₆ branchedalkyne, carboxyl, ester, sulfate, sulfonate, sulfone, amino, ammonium,amido, nitrile, C₁-C₆ alkoxy, phenoxy, substituted phenoxy aromatic,phenyl, polycyclic aromatic, and electron-rich heterocycle.

In some embodiments, R₁ is hydrogen, halogen, methyl, or ethyl. In someembodiments, R₂ and R₃ are each independently a halogen. In someembodiments, R₄ and R₅ are each independently a methyl or ethyl. In someembodiments, R₆ is hydrogen, halogen, methyl, or ethyl. In someembodiments, R₇ is hydrogen, halogen, methyl, ethyl, or SO₃H. In someembodiments, R₈ is hydrogen, halogen, methyl, or ethyl. In someembodiments, R₁ is hydrogen, halogen, methyl, or ethyl; R₂ and R₃ areeach independently a halogen; R₄ and R₅ are each independently a methylor ethyl; R₆ is hydrogen, halogen, methyl, or ethyl; R₇ is hydrogen,halogen, methyl, ethyl, or SO₃H; and R₈ is hydrogen, halogen, methyl, orethyl. In some such embodiments, R₂ and R₃ are each chlorine and R₇ isSO₃H.

In some embodiments, a reference dye is a congener of7-hydroxy-9H-1,3-dichloro-9,9-dimethylacridin-2-one (DDAO). Exemplaryreference dyes include, but are not limited to:

-   1,3-dichloro-7-hydroxy-9,9-dimethylacridin-2(9H)-one (DDAO), and

-   6,8-dichloro-2-hydroxy-9,9-dimethyl-7-oxo-7,9-dihydroacridine-3-sulfonic    acid (Sulfo-DDAO).

Some attributes of reference dyes of formula (I) that are useful for afluorescence reference dye in various methods and compositions of thepresent teachings may include, but are not limited by, high chemical andphotochemical stability, fluorescent emission that is relativelyconstant over a wide temperature range, high water solubility, a broadadsorption spectrum, and a sharp emission spectrum that is spectrallydistinguishable from the reporter dyes used in the assay. Further, anattribute of reference dye of formula (I) may include not interfering inan assay being performed, such as any of a variety of assays utilizingthe polymerase chain reaction (PCR), and variations thereof. Dyes thatmay interfere in an assay may do so, by, for example, either interferingdirectly by inhibiting a reaction, or may interfere by, for example, byinteracting with a probe dye.

The reference dyes of the present teachings may be used in any method inwhich a reference dye is desirable. Exemplary methods in which thereference dyes may be used include, but are not limited to, methodsinvolving polynucleotide detection, methods involving polynucleotideamplification, and methods involving target detection usingoligonucleotides, antibodies, antigens, small molecules,polysaccharides, and the like. Non-limiting exemplary targets include,but are not limited to, polynucleotides, antigens, antibodies,receptors, organelles, membranes, metabolites, enzymes, cells (such aseukaryotic and prokaryotic cells) and the like. Non-limiting exemplarypolynucleotides include DNA and RNA from a variety of nucleicacid-containing samples.

As used herein, the term “nucleic acid-containing sample” refers tonucleic acid found in biological samples according to the presentteachings. It is contemplated that samples may be collected invasivelyor noninvasively. The sample can be on, in, within, from or found inconjunction with a fiber, fabric, cigarette, chewing gum, adhesivematerial, soil or inanimate objects. “Sample” as used herein, is used inits broadest sense and refers to a sample containing a nucleic acid fromwhich a gene target or target polynucleotide may be derived. A samplecan comprise a cell, chromosomes isolated from a cell (e.g., a spread ofmetaphase chromosomes), genomic DNA, RNA, cDNA and the like. Samples canbe of animal or vegetable origins encompassing any organism containingnucleic acid, including, but not limited to, plants, livestock,household pets, and human samples, and can be derived from a pluralityof sources. These sources may include, but are not limited to, wholeblood, hair, blood, urine, tissue biopsy, lymph, bone, bone marrow,tooth, amniotic fluid, hair, skin, semen, anal secretions, vaginalsecretions, perspiration, saliva, buccal swabs, various environmentalsamples (for example, agricultural, water, and soil), research samples,purified samples, and lysed cells. It will be appreciated that nucleicacid samples containing target polynucleotide sequences can be isolatedfrom samples from using any of a variety of sample preparationprocedures known in the art, for example, including the use of suchprocedures as mechanical force, sonication, restriction endonucleasecleavage, or any method known in the art.

The terms “target polynucleotide,” “gene target”, “target genomic locus”and the like as used herein are used interchangeably herein and refer toa particular nucleic acid sequence of interest. Such a target can be apolynucleotide sequence that is sought to be amplified and can exist inthe presence of other nucleic acid molecules or within a larger nucleicacid molecule. The target polynucleotide can be obtained from anysource, and can comprise any number of different compositionalcomponents. For example, the target can be nucleic acid (e.g. DNA orRNA). The target can be methylated, non-methylated, or both. Further, itwill be appreciated that “target” used in the context of a particularnucleic acid sequence of interest additionally refers to surrogatesthereof, for example amplification products, and native sequences. Insome embodiments, a particular nucleic acid sequence of interest is ashort DNA molecule derived from a degraded source, such as can be foundin, for example, but not limited to, forensics samples. A particularnucleic acid sequence of interest of the present teachings can bederived from any of a number of organisms and sources, as recited above.Regarding the ploidy state of a target genomic locus, for an organismwith a diploid genome, in which two alleles define a locus, that thereare three possible genotypes for such a diploid state. One of ordinaryskill in the art will appreciate that any ploidy state is discretelyassociated with a finite number of allelic combinations defining agenotype classification. Thus, for any ploidy state for any samplehaving a target genomic locus of interest, there are a finite andcalculable number of genotypes.

As used herein, “DNA” refers to deoxyribonucleic acid in its variousforms as understood in the art, such as genomic DNA, cDNA, isolatednucleic acid molecules, vector DNA, and chromosomal DNA. “Nucleic acid”refers to DNA or RNA in any form. Examples of isolated nucleic acidmolecules include, but are not limited to, recombinant DNA moleculescontained in a vector, recombinant DNA molecules maintained in aheterologous host cell, partially or substantially purified nucleic acidmolecules, and synthetic DNA molecules. Typically, an “isolated” nucleicacid is free of sequences which naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived.Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule,is generally substantially free of other cellular material or culturemedium when produced by recombinant techniques, or free of chemicalprecursors or other chemicals when chemically synthesized.

In some embodiments of methods and composition of the present teachings,the peak emission wavelength of a reference dye does not overlap withthe peak emission wavelengths of the reporter dyes. The present methodsmay use reference dyes of formula (I), for example, in multiplexreactions. In various embodiments of methods and compositions, whenmultiplexing using spectrally distinct dyes, a reference dye isindependently detectable from the reporter dyes used in the assay.

A multiplex assay may indicate the presence, absence, amount, andidentity of at least two targets in a mixture and comprises at least twoprobes that indicate the presence, absence, amount, and identity of thesame target in a mixture. In some embodiments, a multiplex assay is usedto indicate the presence, absence, amount, and identity of at leastthree, at least four, at least five, or at least six targets in amixture. In various embodiments of methods and composition according tothe present teachings, a multiplex assay may be performed in a samplearray format. In such a multiplex method, multiple targets may bedetected in a single sample region of a sample containment device. Sucha multiplex method would be differentiated from, for example, varioussequencing methods, in which multiplex PCR is performed as a samplepreparation step before sequencing is performed and detected. In variousembodiments of methods and compositions of the present teachings, inwhich a multiplex assay is performed in a sample array format, areference dye of formula (1) may provide for up six or more reporterdyes to be used in such a multiplex assay.

In some embodiments, each of the different targets in the mixture isdetected using a different reporter dye. In some embodiments, the samereporter dye maybe used on two or more different probes to detect thepresence of a single target. In some embodiments, each reporter dye andthe reference dye are detected separately, e.g., by irradiating themixture at each excitation wavelength and detecting the radiationemitted by each reporter dye and the reference dye separately. In someembodiments, two or more of the reporter dyes and the reference dye aredetected simultaneously, either by irradiating with more than oneexcitation wavelength simultaneously and detecting the radiation emittedby more than one of the reporter dyes and the reference dyesimultaneously, or by irradiating with a single excitation wavelengththat is absorbed by more than one of the reporter dyes and the referencedye, and then detecting radiation emitted by more than one of thereporter dyes and the reference dye simultaneously. In some embodiments,one or more of the reporter dyes are fluorescence resonance energytransfer (FRET) dyes. In some such embodiments, for example, when two ofthe reporter dyes are FRET dyes, both dyes may absorb at the samewavelength, but emit at different wavelengths. As will be discussed inmore detail subsequently, selections of spectrally distinct sets ofreporter and reference dyes according to formula (I) may be selected foruse in various embodiments of methods and compositions according to thepresent teachings.

The term “reference dye” as used herein refers to a fluorescent dye offormula (I). In some embodiments, a reference dye is included in amixture and is used to normalize the signals of other dyes (such asreporter dyes) in the assay and mixture. As one of ordinary skill in theart will appreciate, there are many variables in analysis that mayimpact variation of results. A reference dye detection signal mayprovide a reference signal that can be used to adjust the reporter dyedetection signal to correct for fluctuation caused by experimentalvariation, such as pippeting, which may cause variations in sampleconcentration or volume, as well as system variation, such as ofdetection non-uniformity for reporter, reference and background signals,and of thermal non-uniformity. In various embodiments, adjusting ameasured detection signal of a reporter dye may include dividing orsubtracting a measured detection signal of a reference dye from ameasured detection signal of a reporter dye. In various embodiments,adjusting a measured detection signal of a reporter dye may include anymathematical operation in which a measured detection signal of areference dye is used to adjust a measured detection signal of areporter dye.

In some embodiments, a reference dye is included in each mixture of aset of mixtures and may be used to normalize across mixtures. Forexample, in some embodiments, a reference dye may be included in all ofthe wells of a multiwell plate (such as a 96-well plate) and may be usedto normalize the effect of well position on fluorescent signaldetection. In various embodiments of methods and compositions accordingto the present teachings, a reference dye of formula (I) may be used asa “free dye,” meaning that it is not conjugated to another molecule,such as another dye or an oligonucleotide.

The term “reporter dye” as used herein refers to a moiety that is usedin a mixture to indicate the presence, absence, amount, activity, andidentity of a target in the mixture. Reporter dyes may include, but arenot limited to, fluorescent dyes and particles, phosphorescent dyes andparticles, quantum dots, lanthanides, and chemi-luminophores. A reporterdye may be a fluorescence resonance energy transfer (FRET) dye or anon-FRET dye. In some embodiments, a reporter dye may be linked to amoiety selected from an oligonucleotide, a peptide, an antibody, a smallmolecule, and a polysaccharide, wherein the moiety imparts specificityon the reporter dye. In some embodiments, the reporter dye linked to themoiety that imparts specificity is referred to as a “probe.” In someembodiments, the reporter dye indicates the presence, absence, amount,activity, and identity of a target molecule in the assay, wherein themoiety that imparts specificity on the reporter dye is specific for thetarget molecule.

The term “probe,” as used herein, refers to a molecule comprising atarget-specific moiety and at least one reporter dye, which is used todetect the presence, absence, amount, activity, and identity of a targetin a mixture. The target-specific moiety may be linked to at least onereporter dye either covalently or noncovalently. Non-limiting exemplarytarget-specific moieties include oligonucleotides, peptides, antibodies,antigens, small molecules, and polysaccharides. In various embodiments,a probe comprising a peptide and a reporter dye may be referred to as a“labeled peptide;” a probe comprising an antibody and a reporter dye maybe referred to as a “labeled antibody;” a probe comprising an antigenand a reporter dye may be referred to as a “labeled antigen;” a probecomprising a small molecule and a reporter dye may be referred to as a“labeled small molecule;” and a probe comprising a polysaccharide and areporter dye may be referred to as a “labeled polysaccharide.”

As used herein, the terms “amplifying”, “amplification,” and relatedterms refer to any process that increases the amount of a desirednucleic acid. Any of a variety of known amplification procedures may beemployed in the present teachings, including, but not limited to, PCR(see, for example, U.S. Pat. No. 4,683,202), including quantitative andreal-time PCR, as well as any of a variety of ligation-mediatedapproaches, including LDR and LCR (see, for example, U.S. Pat. Nos.5,494,810; 5,830,711; and 6,054,564). Additional non-limitingamplification procedures include, but are not limited to, isothermalapproaches such as rolling circle amplification and helicase-dependantamplification. One of skill in art will readily appreciate a variety ofpossible amplification procedures applicable in the context of thepresent teachings.

In some embodiments, a probe comprises an oligonucleotide and at leastone reporter dye may be used in an amplification reaction. In someembodiments, a probe comprises an oligonucleotide, a reporter dye, and aquencher dye. In some such embodiments, such as a TaqMan® probe,degradation of the oligonucleotide portion of the probe separates thereporter dye from the quencher dye and results in a detectable signalfrom the reporter dye. In some embodiments, a probe comprises twohybridized oligonucleotides, one of which comprises a reporter dye andthe other of which comprises a quencher dye such that when theoligonucleotides are hybridized, the reporter dye signal is quenched. Insome embodiments, such as in the presence of a target that hybridizes toone of the probe oligonucleotides, the reporter dye and the quencher dyeare separated such that a signal is detectable from the reporter dye.

Non-limiting exemplary probes include, but are not limited to, TaqMan®probes (see, e.g., U.S. Pat. No. 5,538,848), stem-loop molecular beacons(see, e.g., U.S. Pat. Nos. 6,103,476 and 5,925,517 and Tyagi and Kramer,1996, Nature Biotechnology 14:303-308), stemless or linear beacons (see,e.g., WO 99/21881), PNA molecular beacons (see, e.g., U.S. Pat. Nos.6,355,421 and 6,593,091), linear PNA beacons (see, e.g., Kubista et al.,2001, SPIE 4264:53-58), non-FRET probes (see, e.g., U.S. Pat. No.6,150,097), Sunrise®/Amplifluor® probes (see, e.g., U.S. Pat. No.6,548,250), stem-loop and duplex Scorpion™ probes (see, e.g., Solinas etal., 2001, Nucleic Acids Research 29:E96 and U.S. Pat. No. 6,589,743),bulge loop probes (see, e.g., U.S. Pat. No. 6,590,091), pseudo knotprobes (see, e.g., U.S. Pat. No. 6,589,250), cyclicons (see, e.g., U.S.Pat. No. 6,383,752), MGB Eclipse™ probes (Epoch Biosciences), hairpinprobes (see, e.g., U.S. Pat. No. 6,596,490), peptide nucleic acid (PNA)light-up probes, self-assembled nanoparticle probes, andferrocene-modified probes. See, e.g., U.S. Pat. No. 6,485,901; Mhlangaet al., 2001, Methods 25:463-471; Whitcombe et al., 1999, NatureBiotechnology. 17:804-807; Isacsson et al., 2000, Molecular Cell Probes.14:321-328; Svanvik et al., 2000, Anal Biochem. 281:26-35; Wolffs etal., 2001, Biotechniques 766:769-771; Tsourkas et al., 2002, NucleicAcids Research. 30:4208-4215; Riccelli et al., 2002, Nucleic AcidsResearch 30:4088-4093; Zhang et al., 2002 Shanghai. 34:329-332; Maxwellet al., 2002, J. Am. Chem. Soc. 124:9606-9612; Broude et al., 2002,Trends Biotechnol. 20:249-56; Huang et al., 2002, Chem Res. Toxicol.15:118-126; and Yu et al., 2001, J. Am. Chem. Soc 14:11155-11161. Insome embodiments, probes comprise black hole quenchers (Biosearch), IowaBlack (IDT), QSY quencher (Molecular Probes), and Dabsyl and Dabcelsulfonate/carboxylate Quenchers and Epoch quenchers. Labeling probes canalso comprise two probes, wherein for example a fluorophore is on oneprobe, and a quencher on the other, wherein hybridization of the twoprobes together on a target quenches the signal, or whereinhybridization on target alters the signal signature via a change influorescence. Labeling probes can also comprise sulfonate derivatives offluorescenin dyes with a sulfonic acid group instead of the carboxylategroup, phosphoramidite forms of fluorescein, phosphoramidite forms of CY5 (available for example from Amersham). In some embodiments,interchelating labels are used such as ethidium bromide, SYBR® Green I(Molecular Probes), and PicoGreen® (Molecular Probes), thereby allowingvisualization in real-time, or end point, of an amplification product inthe absence of a labeling probe.

In some embodiments, a probe comprising an oligonucleotide and areporter dye may be referred to as a “labeled oligonucleotide.” Further,the oligonucleotide portion of the labeled oligonucleotide may be of anylength, such as at least 5, a least 6, at least 7, at least 8, at least9, at least 10, at least 12, at least 15, at least 20, at least 25, atleast 30, at least 35, at least 40, at least 50, at least 60, at least70, at least 80, at least 90, at least 100, at least 120, at least 150,at least 200, etc., nucleotides in length. The oligonucleotide portionof the labeled oligonucleotide may comprise deoxyribonucleic acid (DNA),ribonucleic acid (RNA), peptide nucleic acid (PNA), derivatives of anyof the foregoing, and combinations of any of the foregoing. Regardlessof the composition of the oligonucleotide, each unit of theoligonucleotide that is equivalent to a DNA or RNA base is referred toas a “nucleotide.”

According to various embodiments of methods and compositions of thepresent teachings, a spectrally distinguishable set of reporter dyes anda reference dye according to formula (1) may be used in various assays,such as, but not limited by, various amplification assays, in which athermal cycling instrument may be utilized.

According to various embodiments of a thermal cycler instrument 100, asshown in FIG. 1, a thermal cycling instrument may include a heated cover110 that is placed over a plurality of samples 112 contained in a samplesupport device. Some examples of a sample support device may include,but are not limited to, tubes, vials, and a multi-well plate permittinga selection of sample capacities, such as a standard microtiter 96-well,a 384-well plate. In various embodiments, a sample support device may bea micro device capable of processing thousands of samples per analysis,such as various microfluidic devices, microcard devices, and micro chipdevices. In various embodiments, a sample support device may be afabricated from a substantially planar support, such as a glass, metalor plastic slide, having a plurality of sample regions. The sampleregions in various embodiments of a sample support device may includethrough-holes, depressions, indentations, and ridges, and combinationsthereof, patterned in regular or irregular arrays formed on the surfaceof the substrate. In various embodiments, a sample support device mayhave a cover between the sample regions and heated cover 110. A samplesupport device may have sample regions arranged in a sample arrayformat. One of ordinary skill in the art will recognize that manyexamples of a sample support device are patterned in row and columnarrays. A sample array format according to the present teachings mayinclude any pattern of convenient and addressable arrangement of sampleregions in a sample support device, including a single row or column ofsample regions in a sample support device.

In various embodiments of a thermal cycler instrument 100, include asample block 114, an element or elements for heating and cooling 116,and a heat exchanger 118. Various embodiments of a thermal blockassembly according to the present teachings comprise components 114-118of thermal cycler system 100 of FIG. 1.

In FIG. 2, various embodiments of a thermal cycling system 200 have thecomponents of embodiments of thermal cycling instrument 100, andadditionally a detection system comprising, an imager 210 and optics212. It should be noted that while a thermal cycler system 200 isconfigured to detect signals from samples in a sample support deviceduring an analysis, a detection system according to the presentteachings may be used to detect signals from a thermal cycler system 100after an analysis has been completed.

A detection system may have an electromagnetic radiation source thatemits electromagnetic energy, and a detector or imager 210, forreceiving electromagnetic energy from samples 216 in sample supportdevice. A detector or imager 210 may capable of detectingelectromagnetic energy from samples 216 may a charged coupled device(CCD), back-side thin-cooled CCD, front-side illuminated CCD, a CCDarray, a photodiode, a photodiode array, a photo-multiplier tube (PMT),a PMT array, complimentary metal-oxide semiconductor (CMOS) sensors,CMOS arrays, a charge-injection device (CID), CID arrays, etc. Thedetector can be adapted to relay information to a data collection devicefor storage, correlation, and manipulation of data, for example, acomputer, or other signal processing system. Additionally, optics 212 ofa detection system may include components, such as, but not limited by,various positive and negative lenses, mirrors, and excitation andemission filters.

Regarding various embodiments of an electromagnetic radiation source fora detection system, such sources may include but are not limited to,white light, halogen lamps, lasers, solid state lasers, laser diodes,micro-wire lasers, diode solid state lasers (DSSL), vertical-cavitysurface-emitting lasers (VCSEL), LEDs, phosphor coated LEDs, organicLEDs (OLED), thin-film electroluminescent devices (TFELD),phosphorescent OLEDs (PHOLED), inorganic-organic LEDs, LEDs usingquantum dot technology, LED arrays, an ensemble of LEDs, floodlightsystems using LEDs, and white LEDs, filament lamps, arc lamps, gaslamps, and fluorescent tubes. Light sources can have high radiance, suchas lasers, or low radiance, such as LEDs. The different types of LEDsmentioned above can have a medium to high radiance.

Multiple excitation and emission filter sets can be employed in existingthermal cycling devices, wherein each filter set may includepre-selected excitation and emission filters to provide an accurateresponse of signal proportional to oligonucleotide concentration in asample at various stages of PCR. The excitation filter in a coupled setof filters can be chosen to allow wavelengths of light received from thelight source that are close to the peak excitation wavelength of apredetermined dye to pass. The excitation filter can also be configuredto block wavelengths of light that are greater than and less than thepeak excitation wavelength. Similarly, the emission filter in the set offilters can be chosen to allow light close to the peak emissionwavelength to pass while also blocking wavelengths outside the peakemission wavelength. In such a fashion, and as will be discussed in moredetail subsequently, a selection of spectrally distinguishable dyespecies, in conjunction with the detection system, and data processingcapabilities of a thermal cycling apparatus may provide for detection ofa plurality of dye signals in, for example, a multiplex assay.

In use, a detection system for use with a thermal cycling device mayfunction by impinging an excitation beam from an electromagneticradiation source on samples in a sample support device, therebygenerating a fluorescent radiation from the plurality of samples 116,216. Light emitted from the samples 112, 216, may be transmitted througha lens or lenses, such as a well lens, a Fresnel lens, or a field lens,and then may be directed to additional optical components, such as adichroic mirror, and an emission filter. Undesired wavelengths of lightemitted from the samples 112, 216, may be reflected by the dichroicmirror or are blocked by the emission filter. A portion of the emittedlight that transmits through the dichroic mirror and emission filter maybe received by a detector or imager 210. For a thermal cycler system100, a detector or imager may generate data signals from the fluorescentradiation from the samples at the completion of a PCR assay. For athermal cycler system 200, a detector or imager 210 may generate datasignals from the fluorescent radiation from the samples over time fromwhich the concentration of target DNA in the samples at the particularstage of PCR may be determined.

For embodiments of thermal cycler instrumentation 100 and 200, a controlsystem 120 and 224, respectively, may be used to control the functionsof the detection, heated cover, and thermal block assembly. The controlsystem may be accessible to an end user through user interface 122 ofthermal cycler instrument 100 and 224 of thermal cycler instrument 200.A computer system 300, as depicted in FIG. 3 may serve as to provide thecontrol the function of a thermal cycler instrument, as well as the userinterface function. Additionally, computer system 300 may provide dataprocessing, as well as, with other components, provide for display, andreport preparation functions. For example, signals received by adetector or imager may be processed by various algorithms, such as aspectral deconvolution algorithm, which may then be displayed to an enduser, as well as providing a report. All such instrument controlfunctions may be dedicated locally to the thermal cycler instrument, orcomputer system 300 may provide remote control of part or all of thecontrol, analysis, and reporting functions, as will be discussed in moredetail subsequently.

FIG. 3 is a block diagram that illustrates a computer system 300,according to various embodiments, upon which embodiments of a thermalcycler system 100 of FIG. 1 or a thermal cycler system 200 of FIG. 2 mayutilize. Computer system 300 includes a bus 302 or other communicationmechanism for communicating information, and a processor 304 coupledwith bus 302 for processing information. Computer system 300 alsoincludes a memory 306, which can be a random access memory (RAM) orother dynamic storage device, coupled to bus 302 for instructions to beexecuted by processor 304. Memory 306 also may be used for storingtemporary variables or other intermediate information during executionof instructions to be executed by processor 304. Computer system 300further includes a read only memory (ROM) 308 or other static storagedevice coupled to bus 302 for storing static information andinstructions for processor 304. A storage device 310, such as a magneticdisk or optical disk, is provided and coupled to bus 302 for storinginformation and instructions.

Computer system 300 may be coupled via bus 302 to a display 312, such asa cathode ray tube (CRT) or liquid crystal display (LCD), for displayinginformation to a computer user. An input device 314, includingalphanumeric and other keys, is coupled to bus 302 for communicatinginformation and command selections to processor 304. Another type ofuser input device is cursor control 316, such as a mouse, a trackball orcursor direction keys for communicating direction information andcommand selections to processor 304 and for controlling cursor movementon display 312. This input device typically has two degrees of freedomin two axes, a first axis (e.g., x) and a second axis (e.g., y), thatallows the device to specify positions in a plane. A computer system 300provides data processing and provides a level of confidence for suchdata. Consistent with certain implementations of the present teachings,data processing and confidence values are provided by computer system300 in response to processor 304 executing one or more sequences of oneor more instructions contained in memory 306. Such instructions may beread into memory 306 from another computer-readable medium, such asstorage device 310. Execution of the sequences of instructions containedin memory 306 causes processor 304 to perform the process statesdescribed herein. Alternatively hard-wired circuitry may be used inplace of or in combination with software instructions to implementvarious embodiments of methods and compositions of the presentteachings. Thus implementations of the present teachings are not limitedto any specific combination of hardware circuitry and software.

The term “computer-readable medium” as used herein refers to any mediathat participates in providing instructions to processor 304 forexecution. Such a medium may take many forms, including but not limitedto, non-volatile media, volatile media, and transmission media.Non-volatile media includes, for example, optical or magnetic disks,such as storage device 310. Volatile media includes dynamic memory, suchas memory 306. Transmission media includes coaxial cables, copper wire,and fiber optics, including the wires that comprise bus 302.Transmission media can also take the form of acoustic or light waves,such as those generated during radio-wave and infra-red datacommunications. Common forms of computer-readable media include, forexample, a floppy disk, a flexible disk, hard disk, magnetic tape, orany other magnetic medium, a CD-ROM, any other optical medium, punchcards, paper tape, any other physical medium with patterns of holes, aRAM, PROM, and EPROM, a FLASH-EPROM, any other memory chip or cartridge,a carrier wave as described hereinafter, or any other medium from whicha computer can read.

Further, it should be appreciated that a computer 300 of FIG. 3 may beembodied in any of a number of forms, such as a rack-mounted computer, adesktop computer, a laptop computer, or a tablet computer. According tovarious embodiments of a computer 300 of FIG. 3, a computer may beembedded in any number of mobile and web-based devices not generallyregarded as a computer but with suitable processing capabilities.Example of such devices may include, but are not limited by a PersonalDigital Assistant (PDA), a smart phone, and a notepad or any othersuitable electronic device. Additionally, a computer system can includea conventional network system including a client/server environment andone or more database servers. A number of conventional network systems,including a local area network (LAN) or a wide area network (WAN), andincluding wireless and wired components, are known in the art.Additionally, client/server environments, database servers, and networksare well documented in the art.

As previously discussed, a reference dye selected from members of thefamily of dyes represented by formula (I) below have a number ofdesirable attributes that make them useful candidates as a referencedye:

wherein:

-   -   each of R₁ to R₃ and R₆ to R₈ is independently —H, halogen,        —CO₂H, —CO₂R, —SO₃H, —SO₃R, —CH₂CO₂H, —CH₂CO₂R, —CH₂SO₃H,        —CH₂SO₃R, —CH₂NH₂, —CH₂NHR, —NO₂, C₁-C₆ alkyl, substituted C₁-C₆        alkyl, C₁-C₆ alkoxy, and substituted C₁-C₆ alkoxy, wherein R is        C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, and        substituted C₁-C₆ alkoxy;    -   R₄ and R₅ are either, taken separately, independently selected        from a C₁-C₆ alkyl and a C₁-C₆ substituted alkyl, or, taken        together, are C₃-C₇ cycloalkyl, C₄-C₇ unsaturated cycloalkyl,        C₃-C₇ substituted cycloalkyl, or C₄-C₇ substituted unsaturated        cycloalkyl.

In some embodiments, a reference dye is a congener of7-hydroxy-9H-1,3-dichloro-9,9-dimethylacridin-2-one (DDAO). Exemplaryreference dyes include, but are not limited to:

-   1,3-dichloro-7-hydroxy-9,9-dimethylacridin-2(9H)-one (DDAO) and

-   6,8-dichloro-2-hydroxy-9,9-dimethyl-7-oxo-7,9-dihydroacridine-3-sulfonic    acid (Sulfo-DDAO).

For example, FIG. 4 and FIG. 5 show the normalized emission andexcitation spectra of DDAO and Sulfo-DDAO, respectively. The spectrawere normalized by setting the maximum for each spectrum at 100 unitsprior to plotting. While DDAO and Sulfo-DDAO are exemplified in FIG. 4and FIG. 5, respectively, it is to be understood that any of thereference dyes of formula (I) can be used in the present methods,including, for example, other congeners of DDAO. As shown in FIG. 4 andFIG. 5, the excitation and emission spectra of DDAO and Sulfo-DDAO showthat the reference dyes absorb very long wavelengths, with peakabsorption at about 640 nm. In that regard, DDAO and Sulfo-DDAO alsoemit at very long wavelengths, with peak emission at about 650 to 660nm. In that regard, DDAO, and congeners thereof, such as Sulfo-DDAOviolate the mirror image rule, and have a unique signature in theexcitation and emission spectra. Because the excitation spectrum isbroad and the emission spectrum is compact, DDAO and its congeners, suchas, for example, the compounds of formula (I) are suitable for use witha broad range of excitation source wavelengths as reference dyes in thepresence of reporter dyes, for example, that absorb and emit at shorterwavelengths. Moreover, congeners of DDAO have an emission band thatallows a wider selection of reporter dyes of interest to be spectrallyresolved when dyes of formula (1) are used as a reference dye.

Further, as will be discussed in more detail subsequently, compounds offormula (I) have high water solubility, readily allowing for a range ofconcentrations in aqueous-based solutions and reagents. Additionally,compounds of formula (I) have high chemical and photochemical stability,stable fluorescence emission over a broad range of temperatures, and donot interfere with various biochemical assays, such as PCR. Accordingly,compounds of formula (I) are particularly well suited as reference dyesin bioanalysis.

FIG. 6 depicts a graph of normalized fluorescent emission as a functionof temperature for Sulfo-DDAO in comparison to a reporter dye, 6-FAM, aswell as a reference dye, ROX. Given the difference in brightness of thedyes shown, each data point for each curve was adjusted using aninternal reference point taken from the 94° C. data point for that dataset. In that regard, the fluorescence signals are normalized to giveequal intensities at 94 C so that their relative changes withtemperature can be easily compared. As can be seen from the data, thefluorescence emission is fairly stable across a wide range oftemperatures for Sulfo-DDAO. This attribute may be useful when, forexample, dye calibration may be done at a different temperature from arun temperature, or could be useful for normalizing experimentalvariations as previously discussed.

Regarding chemical stability, DDAO congeners appear to have resilienceunder various conditions, such as oxidation, photo-degradation, and thelike. For example, regarding resilience to harsh oxidation, DDAO,Sulfo-DDAO, and ROX were each suspended as free dyes in a solution of1:4 of Tris-EDTA buffer (1× stock concentration) in MeOH at aconcentration of approximately 1 μM. Five μl of 0.5M benzoyl peroxide inacetonitrile was added to 1 ml of the dye composition in a fluorimetercuvette. Fluorescence emission intensity was measured at each dye'semission maximum immediately, and then at 5 minute intervals. Afirst-order exponential curve was fit to the data to calculate the decayrate for each dye. FIG. 7 is a graph showing the results of thatexperiment. As shown in that figure, both DDAO and Sulfo-DDAO showresilience to oxidation in comparison to ROX. As such, the referencedyes of formula (I) can be used in assays in which the assay componentsmight come in contact with oxidants.

The matrix shown in FIG. 8 was used to test whether Sulfo-DDAO can beused in a genotyping assay as a reference dye without interfering withspectral separation of all possible genotyping signals for a multiplex;in this example, a duplex assay. Four reporter dyes, namely, FAM, VIC,TED, and SID, were used in various mixtures with Sulfo-DDAO as areference dye. Although no sample was used in the assays, theconcentrations of the reporter dyes were controlled to simulate 16different combinations of genotyping signals, as would be collected,processed and displayed by a thermal cycling apparatus. For the mixtureswhere a particular reporter dye was used as a background, the reporterdye was used at a low level as an unquenched dye, to simulate anuncleaved probe signal when combined with the Sulfo-DDAO reference dye.For mixtures where the reporter dye was used to generate a SIGNAL, theconcentration was 10-fold higher compared to the concentration when usedas a background, to simulate a cleaved probe signal. The SIGNALconcentration was meant to mimic a positive test for a specificgenotype.

Data generated from the deconvoluted signals resulting from the variousmixtures shown in FIG. 8 is shown as scatter plots in FIGS. 9A and 9B.The data show good spectral separation for all mixtures, withwell-resolved signals for each positive signal represented by clustersin the upper left and lower right and negative background signalsrepresenting non-template control (NTC) in the lower left cluster. Ineach reaction well the simulated genotype could be correctly called forboth the FAM/VIC genotyping pair and for the TED/SID genotyping pairwhen they are used together in the same assay with Sulfo-DDAO as thepassive reference. Similarly, the experiments outlined in the matrix ofFIG. 8 were done using another reference dye, ROX. FIG. 10B shows signaldetected for a FAM/VIC genotyping pair in the presence of TED/SIDbackground signal (similar to FIG. 10A), but with a ROX passivereference. Poor spectral separation was observed, resulting in falsepositive signals. FIGS. 9A and 9B, in comparison to FIG. 10B demonstratethe impact of selection of an appropriate reference dye; particularly ina multiplex assay, where the reaction matrix is more complex than in anassay where a single probe is used. It should be specifically noted thatthe red-shifted position of the Sulfo-DDAO emission enabled theselection of four spectrally separated reporter dyes; providing for aset of five spectrally distinct dyes.

Additionally, a variety of physico-chemical forces, such as, but notlimited by, Lifschitz-van der Waals, London dispersion, hydrogen, andionic forces, may act to promote dye-dye interactions, such as ringstacking. Such dye-dye interactions may produce untoward results for avariety of assays, such as a variety of assays based on PCR and relatedreactions. The data generated in the model study additionally suggeststhat DDAO congeners do not promote untoward interactions with variousreporter dyes.

As one of ordinary skill in the art is apprised, a master mix is acomposition that may contain almost all of the ingredients required foran amplification reaction except for the sample. The use of a master mixaffords both efficiency and consistency in performing a plurality ofamplification reactions on a plurality of sample regions of a samplesupport device. In various embodiments, a master mix may contain abuffer, a selection of nucleotides, for example, but not limited by,deoxynucleotides (dNTPS i.e. dATP, dGTP, dCTP, and TTP), primers, and atleast one protein moiety. In various embodiments, a master mix maycontain a buffer, a selection of nucleotides, for example, but notlimited by, dNTPS (i.e. dATP, dGTP, dCTP, and TTP), primers, at leastone protein moiety, and a reference dye. In various embodiments, amaster mix may contain a buffer, a selection of nucleotides, forexample, but not limited by, dNTPS (i.e. dATP, dGTP, dCTP, and TTP), andat least one protein moiety. In various embodiments, a master mix maycontain a buffer, a selection of nucleotides, for example, but notlimited by, dNTPS (i.e. dATP, dGTP, dCTP, and TTP), at least one proteinmoiety, and a reference dye. In various embodiments, a master mix may besupplied lyophilized or suspended in a buffer solution.

According to various embodiments of a master mix, a buffer may beselected, for example, but not limited by tris, as well as a variety ofGood's buffers, such as, tricene, bicene, HEPES, and MOPS. In variousembodiments, the buffer may also contain essential non-bufferingingredients, such as various salts, surfactants, and as well as other.Examples of essential non-buffering ingredients may include, but are notlimited by, chloride salts of sodium, potassium, lithium, magnesium andmanganese, as well as surfactants, such as but not limited by,polysorbate surfactants (e.g. Tween 20 and Tween 80), polyoxyethylenesurfactants (e.g. Brij 56 and Brij 58), polyethoxylated phenolsurfactants (e.g. NP-40 and Triton X100), and zwitterionic surfactants(e.g. CHAPS, CHAPSO, Big-CHAP). Additional buffer ingredients mayinclude, for example, but not limited by glycerol, ethylene glycol,propylene glycol, and various molecular weights of polyethylene glycol.According to various embodiments of a master mix, nucleotides may beselected, for example, but not limited by, from dNTPs (i.e. dATP, dGTP,dCTP, and TTP), dideoxynucleotides (didNTPs), deaza-GTP, deaza-dGTP, and2′-deoxyinosine 5′-triphosphate (dITP). For various embodiments of amaster mix, a protein moiety may be selected, for example, but notlimited by, from a DNA polymerase, a ligase, a reverse transcriptase, aribonuclease (e.g. RNase H), a glycosylase (e.g. uracil-N-glycosylase),a single strand binding protein (e.g. GP32), a pyrophosphatase (e.g.from Thermoplasma acidophilum), an albumin, and a gelatin.

FIG. 11 depicts the stability of Sulfo-DDAO in a master mix, andincubated with various additional additives. Sulfo-DDAO was incubated inTaqMan® Universal PCR Master Mix at room temperature for 1 day, 1 week,2 weeks, or 4 weeks. At each designated time point, fixed quantities ofvarious different target polynucleotides and various dye-labeled TaqMan®probes, were added. The target/probe mixtures were PCR amplified in a 96well plate and the signals recorded after each amplification cycle togenerate an amplification curve from which a Ct value was determined foreach target/probe mixture, using Sulfo-DDAO as a reference dye. FIG. 11is a graph showing the results of that experiment, with the Ct valuesplotted for each time point. The Sulfo-DDAO signal was stable at roomtemperature in TaqMan® Universal PCR Master Mix for up to 4 weeks, asevidenced by the consistent Ct values obtained at different incubationtimes for the different target/probe mixtures, using Sulfo-DDAO as areference dye. Further, Sulfo-DDAO did not interfere with theamplification of the various target polynucleotides.

Non-limiting exemplary reporter dyes that can be used, in variousembodiments, in the present methods and kits include any of thosedescribed, for example, in Menchen, et al., U.S. Pat. No. 5,188,934;Benson, et al., U.S. Pat. No. 6,020,481; Lee, et al., U.S. Pat. No.5,847,162; Benson, et al., U.S. Pat. No. 6,008,379; Benson, et al., U.S.Pat. No. 5,936,087; Upadhya, et al., U.S. Pat. No. 6,221,604; Lee, etal., U.S. Pat. No. 6,191,278; Yan, et al., U.S. Pat. No. 6,140,500; Mao,et al., U.S. Pat. No. 6,130,101; Glazer, et al., U.S. Pat. No.5,853,992; Brush, et al., U.S. Pat. No. 5,986,086; Hamilton, et al.,U.S. Pat. No. 6,140,494; Hermann, et al., U.S. Pat. No. 5,750,409;Haugland et al., U.S. Pat. No. 5,248,782; and Karolin et al., JACS 116:7801-6 (1994), each of which is incorporated by reference in itsentirety with regard to fluorescent dye structures, fluorescent dyesynthesis, fluorescent dye conjugation to biopolymers, application offluorescent dyes in energy transfer dyes, and fluorescent dye spectralproperties.

Exemplary reporter dyes also include, but are not limited to,fluorescein dyes such as 5- and 6-carboxyfluorescein (5- and 6-FAM), 5-and 6-carboxy-2′,4′,5′,7′,-4,7-hexachlorofluorescein (5- and 6-HEX®), 5-and 6-carboxy-2′,4′,5′,7′-tetrachlorofluorescein (5- and 6-TET®), NED®,PET®, 1′,2′-benzo-4′-fluoro-7′,4,7-trichloro-6-carboxy-fluorescein,1′,2′,7′,8′-dibenzo-4,7-dichloro-5-carboxyfluorescein. Exemplaryreporter dyes also include, but are not limited to, rhodamine dyes suchas rhodamine green (rhodamine R110), 5-carboxyrhodamine,6-carboxyrhodamine, N,N′-diethyl-2′,7′-dimethyl-5-carboxy-rhodamine(5-R6G), N,N′-diethyl-2′,7′-dimethyl-6-carboxyrhodamine (6-R6G),N,N,N′,N′-tetramethyl-5-carboxyrhodamine (5-TAMRA),N,N,N′,N′-tetramethyl-6-carboxyrhodamine (6-TAMRA),5-carboxy-X-rhodamine (5-ROX), and 6-carboxy-X-rhodamine (6-ROX).Exemplary reporter dyes include, but are not limited to, Alexa dyes suchas Alexa Fluor® 488, Alexa Fluor® 514, Alexa Fluor® 555, Alexa Fluor®568, Alexa Fluor® 594, and Alexa Fluor® 610. Exemplary reporter dyesinclude, but are not limited to, BODIPY dyes such as BODIPY FI, BODIPYR6G, and BODIPY TMR. Exemplary reporter dyes also include, but are notlimited to, quantum dots, FluoroSpheres, and fluorescent microspheres.

Certain on-limiting exemplary reporter dyes are shown in Table 1.

TABLE 1 Absor- Emis- bance sion Extinction Reporter Dye (nm) (nm)Coefficient Pyrene 341 377 43000 7-methoxycoumarin-3-carboxylic acid 358410 26000 ALEXA FLUOR ® 405 401 421 35000 Cascade Blue 399 423 30000AMCA-X (Coumarin) 353 442 19000 ALEXA FLUOR ® 350 346 442 19000 PacificBlue 416 451 37000 Marina Blue 362 459 190007-diethylaminocoumarin-3-carboxylic acid 432 472 56000 EDANS 336 4905700 Syto 9 483 503 65,000 BODIPY 493/503 500 509 79000 BODIPY FL-X 504510 70000 BODIPY FL 504 510 70000 Oregon Green 488 496 516 760006-Carboxyfluorescein (6-FAM) 494 518 80000 Oregon Green 500 499 51984000 ALEXA FLUOR ® 488 495 519 71000 5-Fluorescein 495 520 73000 SybrGreen 494 521 73,000 5-Carboxyfluorescein (5-FAM) 494 522 83000PicoGreen 502 523 Not available Oregon Green 514 506 526 85000 RhodamineGreen-X 503 528 74000 R110 501 529 75300 DY-505 505 530 85000 LuciferYellow 428 532 11000 NBD-X 466 535 22000 BigR110 504 537 115000 dR110520 538 70000 6-Carboxytetrachlorofluorescein (6-TET) 521 538 87000ALEXA FLUOR ® 430 434 541 16000 5(6)-Carboxyeosin 521 544 95000Erythrosin 529 544 90000 BODIPY R6G 528 547 70000 5-R6G 534 549 92300JOE ® 528 550 80000 VIC ® 532 552 97000 6-Carboxyhexachlorofluorescein(6-HEX) 535 553 71000 ALEXA FLUOR ® 532 532 554 81000 5-Carboxyrhodamine6G 524 557 102000 Cascade Yellow 409 558 24000 ALEXA FLUOR ® 555 555 565150000 Big R6G 502 567 80000 548 89000 dR6G 548 567 91200 BODIPY 564/570563 569 142000 BODIPY-TMR-X 544 570 56000 Cy3 552 570 150000 PyMPO 415570 26000 Oyster 556 556 570 155000 NED ® 546 573 69000 Cy3B 558 573130000 ALEXA FLUOR ® 546 556 573 104000 5-Carboxytetramethylrhodamine(5- 546 576 90000 TAMRA) 6-Carboxytetramethylrhodamine (6- 544 576 90000TAMRA) DY-550 553 578 122000 Rhodamine Red-X 560 580 129000 DY-555 555580 100000 6-TAMRA 560 582 97000 TED ® 496 583 89000 568 90900 ABY ® 568583 90900 BODIPY 581/591 581 591 136000 Big TAMRA 499 594 80000 57790000 dTAMRA 578 594 97000 PET ® 558 595 88000 Cy3.5 581 596 1500005-Carboxy-X-Rhodamine (5-ROX) 576 601 82000 Dye 3.5 586 603 124000 TAZ ®499 603 90000 586 124000 Texas Red-X 583 603 116000 ALEXA FLUOR ® 568578 603 91300 6-Carboxy-X-Rhodamine (6-ROX) 587 607 117000 BODIPY TR-X588 616 68000 ALEXA FLUOR ® 594 590 617 73000 dROX 604 617 108000 JUN ®606 618 128900 SID ® 499 618 91100 606 128900 BigROX 501 619 87000 605115000 DY-610 606 636 140000 LIGHTCYCLER ™ Red 640 625 640 BODIPY630/650 625 640 101000 ALEXA FLUOR ® 660 663 690 132000 Cy5.5 675 694250000 DY-675 674 699 110000 DY-676 674 699 84000 ALEXA FLUOR ® 680 679702 184000 LIGHTCYCLER ™ Red 705 685 705 WellRED D3-PA 685 706 224000DY-681 691 708 125000 DY-700 702 723 96000 ALEXA FLUOR ® 700 702 723192000 DY-701 706 731 115000 DY-730 734 750 113000 WellRED D2-PA 750 770170000 ALEXA FLUOR ® 750 749 775 240000 DY-750 747 776 45700 DY-751 751779 220000 DY-782 782 800 102000

As one skilled in the art will appreciate, a set of dyes can beconfigured to include a passive reference dye according to the presentteachings and one or more of the many dyes listed in Table 1 as reporterdyes, to achieve a plurality of dyes that are each independentlyspectrally distinguishable from the other dyes of the set. One skilledin the art can select a suitable set of reporter dyes and at least onereference dye of formula (I), according to the intended application. Oneor more of the selected reporter dyes may be from the lists and patentsdiscussed herein, or may be any other reporter dyes known in the art. Insome embodiments, a spectrally distinguishable set of reporter dyes ischosen. In some embodiments, each dye in an assay has a peak emissionwavelength that may be at least 5 nm, at least 10 nm, at least 15 nm, atleast 20 nm, or at least 30 nm from the peak emission wavelength of anyof the other dyes in the assay. As previously discussed, the spectralseparation of the dyes, in conjunction with various embodiments of adetection system and computational means allowing for data processingmay together provide for various analyses, such as multiplexing.

Various embodiments of methods and compositions according to the presentteachings comprise measuring fluorescence emitted by the reference dye.In some embodiments, the method comprises measuring fluorescence emittedby at least one reporter dye. In some such embodiments, the fluorescencemay be emitted under excitation conditions suitable for the dye whosefluorescence is being measured. In some embodiments, the method furthercomprises normalizing the measured fluorescence emitted by at least onereporter dye based on the measured fluorescence emitted by the referencedye, to form a normalized measurement. The normalized measurement foreach reporter dye can then be compared to the normalized measurement forone or more other reporter dyes in the reaction. Further, in someembodiments, one or more of the normalized measurements can be printedout, displayed, stored, or otherwise manipulated.

In some embodiments, a reaction mixture for an assay may comprise atleast one, at least two, at least three, at least four, at least five,or at least six different reporter dyes that are each spectrallyseparated from one another, as well as a reference dye. In someembodiments, the reaction mixture may comprise at least one, at leasttwo, at least three, at least four, at least five, or at least sixdifferent probes. In various embodiments, the mixture comprises one tosix, one to five, one to four, two to six, two to five, two to four,three to six, three to five, or three to four distinct probes. In someembodiments, a probe is a labeled oligonucleotide. In some embodiments,a probe is a labeled peptide, a labeled antibody, a labeled antigen, alabeled small molecule, or a labeled polysaccharide.

In some embodiments, the method comprises irradiating the mixture with afirst excitation wavelength and detecting light emitted from at least afirst reporter dye. In some embodiments, the method comprisesirradiating the mixture with a second excitation wavelength, anddetecting light emitted from at least a second reporter dye. In someembodiments, the second excitation wavelength differs from the firstexcitation wavelength. In some embodiments, a different excitationwavelength is used for each different reporter dye in the mixture, andfor the reference dye. In some embodiments, the same excitationwavelength may be used for more than one reporter dye and reference dyein the mixture, although each of the more than one reporter dye andreference dye in the mixture can be distinguished by their emissions.That is, in some embodiments, multiple excitation wavelengths may beused simultaneously to excite more than one reporter dye and referencedye at the same time. Further, in some embodiments, followingexcitation, while the emission spectra of more than one reporter dye inthe mixture may overlap, there is at least one wavelength for each dyeat which that dye is the predominant emitter and therefore that dye isspectrally distinct, and can be detected separately from the others atleast that wavelength.

In some embodiments, one or more excitation wavelengths may be generatedfrom an electromagnetic radiation source. In some embodiments, such anelectromagnetic radiation source may be used to excite one or morereporter dyes and reference dyes. The method comprises, in someembodiments, actuating a broad wavelength emitting source and spectrallyseparating excitation beams from the light source to form at least twoexcitation sources of at least two different respective excitationwavelength ranges. In some embodiments, for simultaneous excitation oftwo or more dyes, the method can comprise forming at least twodifferent, non-overlapping excitation wavelength ranges, at the sametime.

In some embodiments, each dye can emit light, upon excitation, within arespective peak emission wavelength range, for example, having a widthof about 10 nm centered at a peak emission wavelength of the dye. Insome embodiments, each dye emits light within a respective peak emissionwavelength range having a width in a range of about 5 nm-30 nm centeredat a peak emission wavelength of the dye. The dyes can be selected, insome embodiments, such that the respective peak emission wavelength ofeach dye does not overlap with the peak emission wavelength of any ofthe other dyes in the mixture. The dyes can be selected, in someembodiments, such that the respective emission decay rates of each dyeis different from any of the other dyes in the mixture. To distinguishthe emission of the reference dye from the emissions of the reporterdyes, in some embodiments, the fluorescence generated by the referencedye can be filtered, using a first optical filter, and the fluorescencegenerated by at least one reporter dye can be filtered using a secondoptical filter that differs in band pass from the first optical filter.In some embodiments, the first optical filter and the second opticalfilter can together comprise a single filter.

Exemplary sets of spectrally distinguishable reporter dyes, with which areference dye of formula (I) may be used, include, but are not limitedto, 6-FAM™, VIC®, TED®, TAZ®, and SID®; 6-FAM®, VIC®, ABY®, JUN®, andDye 3.5; 6-FAM, TET®, HEX®, ABY®, Dye 3.5, and JUN®; and 6-FAM™, VIC®,NED®, and PET®. In some embodiments, each dye of the plurality of dyescan emit radiation, upon excitation, within a respective peak emissionwavelength range. The dyes can be selected such that they are spectrallydistinguished so that the respective peak emission wavelength range ofeach dye does not overlap with the peak emission wavelength range of anyof the other dyes of the plurality. In some embodiments, one or more ofthe reporter dyes have a peak emission wavelength that is shorter thanthe peak emission wavelength of the reference dye. In some embodiments,one or more of the reporter dyes have a peak emission wavelength that islonger than the peak emission wavelength of the reference dye. For thepurpose of illustration, non-limiting examples are given in FIG. 12-16.

FIG. 12 shows an exemplary normalized emission spectrum of each dye in aon-limiting exemplary set of four reporter dyes with Sulfo-DDAO as areference dye. The four reporter dyes are 6-FAM™, VIC®, NED®, and PET®,which have peak emissions of 518, 552, 573, and 595 nm, respectively.Each dye is conjugated to an oligonucleotide, and the spectra are takenat 1 μM in Tris-EDTA buffer (1× stock concentration). The reference dyeis Sulfo-DDAO, and is not conjugated to an oligonucleotide. FIG. 12shows an exemplary peak emission separation that can be achieved betweena suitable set of reported dyes and the reference dye. Other setsreporter dyes can be chosen from the many possibilities described hereinand known in the art.

FIG. 13 shows the normalized emission intensity of each dye of a set ofdyes according to various embodiments of the present teachings, whereinthe set includes Sulfo-DDAO as a passive reference dye and four reporterdyes of shorter emission wavelengths. The four reporter dyes are 6-FAM™,VIC®, TED®, and SID®, which have peak emissions of 518, 552, 583, and618 nm, respectively. Each dye is conjugated to an oligonucleotide, andthe spectra are taken at 1 μM in Tris-EDTA buffer (1× stockconcentration). The reference dye is Sulfo-DDAO, is a free dye and isnot conjugated to an oligonucleotide. FIG. 13 shows an exemplary peakemission separation that can be achieved between a suitable set ofreported dyes and the reference dye. Similarly, FIG. 14 shows a graph ofnormalized emission intensity for a set of dyes according to variousembodiments of the present teachings, wherein the set comprisesSulfo-DDAO as a passive reference dye and four reporter dyes of shorterwavelengths. The four oligonucleotide-bound reporter dyes shown are6-FAM™, VIC®, ABY®, and JUN®, which have peak emissions of 518, 552,583, and 618 nm, respectively. Other sets reporter dyes can be chosenfrom the many possibilities described herein and known in the art.

FIG. 15 is a graph showing the normalized emission intensity of each dyeof a set of six dyes according to various embodiments of the presentteachings, wherein the set includes Sulfo-DDAO as a passive referencedye and five reporter dyes of shorter wavelengths In variousembodiments, and as shown in FIGS. 12-14, the reporter dyes can beselected so that the set is spectrally distinct and readilyresolvability from other dyes of the set with respect to detection. Thefive reporter dyes shown are 6-FAM™, VIC®, ABY®, Dye 3.5, and JUN®,which have peak emissions of 518, 552, 583, 603, and 618 nm,respectively. Each dye is conjugated to an oligonucleotide, and thespectra are taken at 1 μM in Tris-EDTA buffer (1× stock concentration).The reference dye is Sulfo-DDAO is a free dye, and is not conjugated toan oligonucleotide. Other sets reporter dyes can be chosen from the manypossibilities described herein and known in the art.

FIG. 16 is a graph showing the normalized emission intensity of each dyeof a set of seven dyes according to various embodiments of the presentteachings, wherein the set includes Sulfo-DDAO as a passive referencedye and six active days of shorter wavelengths. The reporter dyes can beselected so that the set is spectrally distinct and readilyresolvability from other dyes of the set with respect to detection. Thesix reporter dyes shown are 6-FAM™, TET®, HEX®, ABY®, Dye 3.5, and JUN®,which have peak emissions of 518, 538, 553, 583, 603, and 618 nm,respectively. Each dye is conjugated to an oligonucleotide, and thespectra are taken at 1 μM in Tris-EDTA buffer (1× stock concentration).The reference dye is Sulfo-DDAO is a free dye, and is not conjugated toan oligonucleotide. Other sets reporter dyes can be chosen from the manypossibilities described herein and known in the art.

FIG. 17 is a matrix showing the results of SNP genotyping of 12different loci in corn using Sulfo-DDAO as a reference dye in afour-reporter TAQMAN® Duplex experiment. Two allele-specific probes foreach locus are labeled with FAM™ and VIC® (a) or TED® and SID® (b) andassayed together. The genomic DNA and four probes were suspended in amultiplex PCR master mix in a 96 well plate and subjected to 40 cyclesof PCR prior to measuring the end-point fluorescence signal in eachwell. In that experiment, about 98% of the reaction sets yielded goodgenotyping results. That is, for 98% of the assay combinations, two lociwere able to be genotyped in the same assay without interference fromthe other. Only the two combinations shown in light gray yielded lessthan optimal results (10a/9b and 11a/9b). None of the reactions failedto amplify the targets, however. Accordingly, these results demonstratethat Sulfo-DDAO can be used as a reference dye in multiplexed systems,such as this four-reporter system.

Variations of kit components are contemplated in various embodiments ofa kit according to the present teachings. In some embodiments of a kit,a probe is a labeled oligonucleotide, which comprises an oligonucleotideand a reporter dye. In some embodiments, the reporter dye is afluorescent dye. In some embodiments, the reporter dye may be selectedfrom the fluorescent dyes listed in Table 1. In some embodiments, thereference dye may be an ingredient in a master mix in a kit. In someembodiments, the reference dye and a master mix maybe separatecontainers in a kit. In some embodiments, the reference dye and each ofthe at least one probes may be in separate containers in a kit. In someembodiments, the reference dye may be in a master mix, and the mastermix and each of the at least one probes are in separate containers in akit. In some embodiments, the reference dye, a master mix, and each ofthe at least one probes may be in separate containers in a kit. In someembodiments, the reference dye and at least one of the probes may be inthe same container in the kit. In some embodiments, at least two of theprobes may be in the same container, while the reference dye is in aseparate container in the kit. In various embodiments, the kitcomponents may be supplied lyophilized or suspended in a buffersolution. In some embodiments, the kit comprises instructions for usingthe reference dye and the probes. Such instructions, in someembodiments, may be instructions for carrying out an assay.

Reporter dyes that can be used in the present teachings are not limitedto those discussed herein and can include a wide variety of differentdyes, including, but not limited to, FRET dyes, non-FRET dyes, andcombinations thereof. As noted above, in some embodiments, the reporterdyes are spectrally distinguishable from each other and from thereference dye. In some embodiments, the reporter dyes aredistinguishable from each other and from the reference dye by theiremission decay rates. The reporters can be chosen from the manypossibilities discussed herein and in Table 1 above. The ability to usea stable reference dye with a set of six other dyes as shown in FIG. 16is evidence that the Sulfo-DDAO reference dye of the present teachingscan be useful in multiplexing assays that can detect, for example, butnot limited by, at least six different analytes in the same assay.Different emission filter band passes, filter wheels, and otherfiltering and detection systems that can be used in such a methodinclude those described herein and those that will become apparent tothose of skill in the art given the present teachings.

While the present teachings have been described in conjunction withvarious embodiments and examples, it is not intended that the presentteachings be limited to such embodiments or examples. On the contrary,the present teachings encompass various alternatives, modifications, andequivalents, as will be appreciated by those of skill in the art. Theteachings should not be read as limited to the described order orelements unless stated to that effect. It should be understood thatvarious changes in form and detail may be made without departing fromthe scope of the present teachings, including the order and arrangementof disclosed method steps. Therefore, all embodiments that come withinthe scope and spirit of the present teachings and equivalents theretoare claimed.

What is claimed is:
 1. A multiplex assay technique comprising (a)combining in a mixture: at least two different targets, at least twoprobes each comprising a target-specific moiety, a reporter dye, and aquencher dye, wherein each target-specific moiety is an oligonucleotideand is specific for a different target and each reporter dye isdifferent from the other reporter dyes and is different from thereference dye, and a reference dye having a structure according toformula (I):

wherein: each of R₁ to R₃ and R₆ to R₈ is independently —H, halogen,—CO₂H, —CO₂R, —SO₃H, —SO₃R, —CH₂CO₂H, —CH₂CO₂R, —CH₂SO₃H, —CH₂SO₃R,—CH₂NH₂, —CH₂NHR, —NO₂, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆alkoxy, and substituted C₁-C₆ alkoxy, wherein R is C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, and substituted C₁-C₆ alkoxy; R₄and R₅ taken separately are selected from C₁-C₆ alkyl, and C₁-C₆substituted alkyl, or R₄ and R₅ taken together are selected from C₃-C₇cycloalkyl, C₄-C₇ unsaturated cycloalkyl, C₃-C₇ substituted cycloalkyl,or C₄-C₇ substituted unsaturated cycloalkyl; (b) amplifying the mixtureof (a) to form an amplified mixture; (c) irradiating the mixture with afirst excitation wave length; and (d) detecting the radiation emitted bythe at least two reporter dyes, which can each be measured in thepresence of the other reporter dyes and the reference dye; wherein theassay indicates the presence, absence, amount, and/or identity of the atleast two different targets in the mixture.
 2. The multiplex assaytechnique of claim 1, wherein at least one reporter dye is afluorescence resonance energy transfer (FRET) dye.
 3. The multiplexassay technique of claim 1, wherein in step f) the amplified mixture isirradiated with a second excitation wave length.
 4. The multiplex assaytechnique of claim 1, wherein the mixture comprises at least threedifferent targets and at least three different probes, and wherein theassay indicates the presence, absence, amount, and/or identity of the atleast three different targets in the mixture.
 5. The multiplex assaytechnique of claim 4, wherein the mixture comprises at least fourdifferent targets and at least four different probes, and wherein theassay indicates the presence, absence, amount, and/or identity of the atleast four different targets in the mixture.
 6. The multiplex assaytechnique of claim 5, wherein the mixture comprises at least fivedifferent targets and at least five different probes, and wherein theassay indicates the presence, absence, amount, and/or identity of the atleast five different targets in the mixture.
 7. The multiplex assaytechnique of claim 6, wherein the mixture comprises at least sixdifferent targets and at least six different probes, and wherein theassay indicates the presence, absence, amount, and/or identity of the atleast six different targets in the mixture.
 8. The multiplex assaytechnique of claim 1, wherein R₁ is selected from hydrogen, halogen,methyl, and ethyl; R₂ and R₃ are each independently a halogen; R₄ and R₅are each independently selected from methyl and ethyl; R₆ is selectedfrom hydrogen, halogen, methyl, and ethyl; R₇ is selected from hydrogen,halogen, methyl, ethyl, and SO₃H; and R₈ is selected from hydrogen,halogen, methyl, and ethyl.
 9. The multiplex assay technique of claim 8,wherein R₂ and R₃ are each chlorine and R₇ is hydrogen or SO₃H.
 10. Themultiplex assay technique of claim 1, wherein the reference dye isselected from: